The proposed program intends to discover antiviral compounds that target functional RNA components of the hepatitis C virus (HCV) genome. The specific aims of this project are to: 1) define subdomains of functional RNA elements in the HCV genome that are amenable to biochemical and biophysical characterization;2) assess subdomains by RNA-motif analysis for the potential to contain ligand-binding sites;3) prioritize RNA subdomains for further investigation by assessment of potential ligand binding sites and published biological data;4) develop oligonucleotide model systems for biochemical and biophysical characterization as well as crystallization of RNA subdomains;5) develop RNA affinity assays for the HCV subdomains;6) determine the three-dimensional structure of RNA subdomains by X-ray crystallography;7) design and synthesize novel RNA-biased ligands based on two chemical classes of RNA-"friendly" compounds;8) identify ligands that bind to selected HCV RNA subdomains by using affinity assays;9) test the positive binders for their target specificity;10) test ligands for their interference with viral translation by developing and applying an HCV IRES-driven in vitro translation assay;11) test translation inhibitors for permeability in mammalian cells;12) test translation inhibitors for inhibition of viral replication in mammalian cells;13) determine the three-dimensional structure of RNA-ligand complexes by crystallography;14) design modified ligands with potentially improved binding affinity by using structural information. The lack of a vaccine and direct antiviral drugs to treat or prevent the spread of HCV creates an urgent need for the development of new therapeutics. The viral RNA is an attractive target for small molecules that recognize structured functional domains of the HCV genome and interfere with protein synthesis. Rational structure-guided design along with synthetic chemistry of RNA-"friendly" compounds will facilitate the generation of RNA-binding molecules that display specific target recognition and biological activity against HCV protein synthesis. This research is aimed at the discovery of new classes of molecules that will significantly advance the development of potent antiviral drugs for combating HCV infection. Such advances are critical for the future ability of humanity to defeat viral diseases.